Neural progenitor–derived Apelin controls tip cell behavior and vascular patterning

During angiogenesis, vascular tip cells guide nascent vascular sprouts to form a vascular network. Apelin, an agonist of the G protein–coupled receptor Aplnr, is enriched in vascular tip cells, and it is hypothesized that vascular-derived Apelin regulates sprouting angiogenesis. We identify an apelin-expressing neural progenitor cell population in the dorsal neural tube. Vascular tip cells exhibit directed elongation and migration toward and along the apelin-expressing neural progenitor cells. Notably, restoration of neural but not vascular apelin expression in apelin mutants remedies the angiogenic defects of mutants. By functional analyses, we show the requirement of Apelin signaling for tip cell behaviors, like filopodia formation and cell elongation. Through genetic interaction studies and analysis of transgenic activity reporters, we identify Apelin signaling as a modulator of phosphoinositide 3-kinase and extracellular signal–regulated kinase signaling in tip cells in vivo. Our results suggest a previously unidentified neurovascular cross-talk mediated by Apelin signaling that is important for tip cell function during sprouting angiogenesis.


INTRODUCTION
To establish a three-dimensional vascular network that sustains organ function, endothelial cells (ECs) leave preexisting vessels and migrate into avascular areas in a process termed sprouting angiogenesis.Vascular endothelial growth factor (VEGF) signaling initiates angiogenesis by triggering individual ECs to sprout from neighboring vessels.Subsequently, trailing cells follow leading cells, resulting in the formation of a sprout with specialized tip cells at the forefront and stalk cells trailing behind (1).Within the vascular sprouts, ECs dynamically compete for the tip cell position (2).Tip cells, characterized by dynamic filopodia extensions, sense secreted molecules such as VEGF-A (1,3).Zebrafish embryos deficient for vegfaa or the Vegfr2 paralog kdrl exhibit severe defects of intersegmental vessel (ISV) outgrowth, with delayed budding of tip cells and absence of most ISVs (4,5).Increased VEGF signaling levels in tip cells lead to elevated extracellular signal-regulated kinase (ERK) activity and DLL4 (Delta-like 4) expression, which inhibits tip cell fate in stalk cells through Notch receptor signaling (6)(7)(8)(9).In addition, asymmetric cell divisions resulting in larger tip daughter cells compared to stalk daughter cells maintain high VEGF signaling in tip cells (10).
While recent studies have elucidated the importance of the Apelin signaling pathway in angiogenesis across various species and in different contexts (11)(12)(13)(14)(15)(16)(17)(18)(19), the specific mechanisms governing its regulation of tip cell behavior and migration remain unknown.The Apelin receptor (Aplnr), a G protein-coupled receptor (GPCR), is expressed in angiogenic ECs (11), whereas its ligand Apelin (Apln) is enriched in tip cells (11,20).In zebrafish, the Apelin signaling pathway is composed of two ligands, Apela (also known as Elabela or Toddler) and Apln, along with two receptors Aplnra and Aplnrb.Previously, it has been shown that Apela is expressed in the early embryo and regulates mesodermal cell movements (21) and angioblast migration (15).Yet, our previous findings show that the development of the ISVs mainly depends on Apln and Aplnrb (11).Moreover, zebrafish mutants for apln or aplnrb exhibit defects in the morphology of tip cells and their migration (11).However, the exact molecular and cellular mechanisms by which Apelin signaling regulates endothelial tip cell behavior and migration are not known.
In this study, we use state-of-the-art confocal time-lapse imaging of developing zebrafish embryos, coupled with novel transgenic reporters and genetic experiments, to unravel a previously undiscovered neurovascular cross-talk mediated by Apelin signaling.Our findings reveal that Apelin signals originate from neural progenitor cells in the dorsal neural tube and are transmitted to the tip cell.Neural progenitor-derived Apelin guides tip cell migration toward the neural tube, facilitating the formation of the dorsal longitudinal anastomotic vessel (DLAV) by directing migration of tip cells along apln-expressing cells.This paracrine signaling regulates tip cell specific behaviors such as filopodia formation, asymmetric daughter cell size after division, and tip-stalk cell shuffling.Furthermore, by using genetic interaction experiments and transgenic biosensors, we found that Apelin signaling exerts its regulatory effects through the activation of phosphoinositide 3-kinase (PI3K) and ERK signaling pathways.

During ISV sprouting, apelin is predominantly expressed in a subpopulation of neural progenitor cells in the dorsal neural tube
To visualize apln expression at a single-cell resolution, we previously generated a transgenic bacterial artificial chromosome (BAC) reporter driving green fluorescent protein (GFP) expression under the control of the apln promoter that allowed us to confirm expression of apln in tip cells of ISVs (11).However, the long half-life of GFP (~26 hours) (22) does not allow to monitor transcriptional changes over time.To accurately monitor transcriptional changes over time, we developed a new reporter and replaced GFP with Venus-PEST, which has a shorter half-life (23).Confocal time-lapse imaging was performed on TgBAC(apln:Venus-PEST); Tg(kdrl:HsHRAS-mCherry) transgenic zebrafish embryos between 26 and 48 hours after fertilization (hpf ) to track apln:Venus-PEST expression (Fig. 1A and movie S1).To our surprise, we observed that apln:Venus-PEST expression was highest in cells located in the dorsal neural tube at 26 and 32 hpf and its expression in the vasculature became apparent only after the formation of the DLAV at 40 hpf.Notably, we found that the tip cells extended dorsally toward the apln: Venus-PEST-expressing cells at 26 hpf (Fig. 1A).Once the tip cells reach the dorsal side of the embryo, they begin to form a T-shape, connect, and establish the DLAV (24).We observed that tip cells migrate along apln:Venus-PEST-expressing cells and maintained close contact (Fig. 1, A and C, and fig.S1A).At 48 hpf, apln:Venus-PEST expression was strongest in the ECs of the DLAV, while apln:Venus-PEST expression in the neural tube decreased (Fig. 1A and fig.S1A).We found that the novel TgBAC(apln:Venus-PEST) reporter (Fig. 1A and fig.S1, A and B) reflects endogenous apln expression, as determined by in situ hybridization (fig.S1, B and C).However, while we could not detect apln:Venus-PEST expression in tip cells before 32 hpf, faint apln mRNA expression in tip cells was detectable by in situ hybridization at 24 hpf (Fig. 1A and fig.S1C).
This discrepancy between the endogenous expression and the reporter expression could be due to the delay between the presence of the mRNA and folding of the fluorophore.In addition, we detected aplnrb expression predominantly in the vasculature between 24 and 48 hpf (Fig. 1B and fig.S1C).
Considering that radial glial cells represent a substantial proportion of cells in the developing zebrafish neural tube (25), we investigated the expression of apln in radial glia cells by using the [TgBAC(gfap:GAL4FF); Tg(UAS-E1B:NTR-mCherry)] reporter line (Fig. 2A).We observed coexpression of apln:Venus-PEST with gfap:GAL4FF; UAS-E1B:NTR-mCherry at 26 hpf, and at 56 hpf, all cells positive for Venus-PEST in the neural tube were positive for the mCherry (Fig. 2A).To further characterize the cell population in the neural tube that express apln, we analyzed optical transverse sections (fig.S3A).We observed apln-expressing cells in a cell population limited to the dorsal part of the neural tube (fig.S3, A and B) and that most of the cells exhibit characteristics of (radial) glial cells (fig.S3C), while fewer cells express neuronal markers (fig.S2B).Therefore, we hypothesized that these apln-expressing cells could be progenitor cells, capable of developing into different cell types.To test this hypothesis, we assessed if the apln + cells express the well-established neural progenitor cell marker Nestin (Nes) (26).Upon injecting of a plasmid expressing mScarlet-I3 under the control of the nes promoter, we could detect double apln + nes + positive cells at 52 hpf (fig.S4A).However, most cells that strongly express nes:mScarlet-I3 localize ventrally to the apln: Venus-PEST-expressing cells, which is in line to what we observed in the TgBAC(nes:EGFP) transgenic line (fig.S4, A and B).Because we observed that the apln-expressing cells are located in the dorsal part of the neural tube, we analyzed if these cells are part of the roof plate.To test this, we analyzed the expression of zic2b, a wellknown marker for roof plate cells, as well as dorsal progenitors, interneurons, and cranial neural crest domains (27)(28)(29)(30)(31). Therefore, we generated two novel transgenic reporter lines for zic2b [Tg(zic2b: mCherry), Tg(zic2b:GAL4-VP16)].By analyzing Tg(zic2b:mCherry); TgBAC(apln:Venus-PEST) double transgenic embryos, we could detect zic2b:mCherry expression in the dorsal neural tube at 26, 32, and 48 hpf (Fig. 2B).Notably, we observed that the apln:Venus-PEST + cells express zic2b:mCherry and localize ventrally to the cells that displayed the highest zic2b:mCherry expression (Fig. 2B).Further analysis revealed that the Tg(zic2b:mCherry) reporter marks interneurons (fig.S5A, neural tube margin) and roof plate cells (fig.S5, A to C, central neural tube) (28,32).We observed axonallike projections emerging from cells expressing apln:Venus-PEST and elavl3:GAL4-VP16; UAS-E1B:NTR-mCherry or zic2b:GAL4-VP16; UAS-E1B:NTR-mCherry (fig.S6, A to D, arrows).Notably, the expression of apln:Venus-PEST in these cells rapidly declines once the projection is formed (fig.S6, A and B, arrows).Considering their position within the neural tube (figs.S2 to S5), their distinct morphology, characterized by central cell bodies with projections extending outward of the apln:Venus-PEST-expressing cells (fig.S3C), as well as their expression of zic2b (fig.S5, A and B), we propose that these cells are dorsal neural progenitor cells, which can give rise to interneurons.To trace the fate of the apln-expressing cells over time, we performed inducible genetic lineage tracing using TgBAC(apln:Cre-ERT2); Tg(−3.5ubb:loxP-EGFP-loxP-mCherry);Tg(kdrl:TagBFP) transgenic embryos and induced recombination from 25 to 30 hpf by adding 4-Hydroxytamoxifen (4-OHT) to water (Fig. 2C).Upon 4-OHT treatment, apln:Cre-ERT2-expressing cells and their descendants are permanently labeled with mCherry.At 120 hpf, we identified mCherry-labeled cells in the dorsal half of the neural tube as well as mCherry-labeled ECs (Fig. 2, C and C′, arrowheads).Among the mCherry-labeled cells, we identified cell types with the characteristic morphology of neurons (Fig. 2C″, asterisks), radial glial cells (Fig. 2C″, arrows), and roof plate progenitor cells (Fig. 2C″, arrowheads, and movie S2), confirming our earlier observations.In conclusion, our data indicate that apln is transiently expressed (24 to 56 hpf) in dorsal neural progenitor cells in the zebrafish neural tube.After 32 hpf, apln expression in dorsal neural progenitor cells declines, likely caused by the differentiation of the progenitor cells.Simultaneously, vascular apln expression in the DLAV increases from 32 hpf onward.
Neural progenitor cell-derived Apelin regulates tip cell migration and DLAV formation apln is known to be expressed in vascular tip cells (11,20).Our discovery that apln is expressed in dorsal neural progenitor cells during ISV sprouting led us to investigate the respective contributions of neural and vascular Apln in the regulation of sprouting angiogenesis (Fig. 3A).First, we tested if Apln gradients attract tip cells toward the neural tube.Overall, our data show that tip cells in the ISV require a source of Apelin from the neural tube to form the T-shape and anastomose to form the DLAV.In contrast, dorsal migration of tip cells toward the neural tube is less dependent on Apelin from the neural tube.

Apelin signaling drives the elongation of tip cells
On the basis of our findings, we aimed to characterize the impact of neural progenitor cell-derived apln on tip cell behavior and performed

high-resolution time-lapse imaging of the actin cytoskeleton of tip cells by analyzing Tg(fli1a:GAL4FF); Tg(UAS:LIFEACT-EGFP);
Tg(kdrl:NLS-mCherry) embryos (Fig. 4A).We found that tip cells exhibit four distinct morphological changes during ISV sprouting (Fig. 4A): (i) Initially, tip cells exhibit a compact morphology; (ii) formation of a long dorsal filopodium after crossing the horizontal myoseptum; (iii) elongation of the cell body while the nucleus remained stationary; and (iv) formation of T-shaped connections with neighboring ISV tip cells, accompanied by dorsal movement of the nucleus.
To improve visualization of dynamic filopodia formation, we generated a novel transgenic line [Tg(kdrl:EGFP-CAAX)] expressing membrane-bound GFP under the control of the kdrl promoter, providing a higher spatial resolution of membrane dynamics (Fig. 4, B and C).
Consistent with previous findings, we observed that tip cells formed one or two dominant filopodia (3), which preceded tip cell elongation (Fig. 4B).To investigate the role of Apelin signaling on tip cell behavior at a single-cell resolution, we analyzed aplnrb mutant embryos from 25 hpf onward (Fig. 4C and movie S3).The initial phenotype of aplnrb mutant tip cells is not markedly different from that of wild-type tip cells, although migration was slower in aplnrb mutant tip cells (Fig. 4C, 0 min).However, as wild-type tip cells reached the dorsal edge of the notochord/horizontal myoseptum and start to elongate (Fig. 4, C and E), aplnrb mutant tip cells fail to form a dominant long filopodium (Fig. 4C) and did not elongate (Fig. 4E).By analyzing filopodia formation, we observed that filopodia from control embryos are longer when extending toward the dorsal side [45° to 90° relative to the dorsal aorta (DA)] than toward the lateral side (0° to 45° relative to the DA) (mean dorsal, 16.54 μm; lateral, 14.61 μm) (Fig. 4D).However, aplnrb mutant embryos exhibited a reduction in filopodia length in both dorsal and lateral directions (mean dorsal, 7.65 μm; lateral, 7.48 μm) (Fig. 4D).
The lengths of filopodia in aplnrb mutants is nearly identical in both directions, and filopodia longer than 20 μm are completely absent (Fig. 4D).To investigate if Apelin signaling modulates actin dynamics in tip cells, we treated embryos with latrunculin B to block actin polymerization (33).Treatment with 375 nM latrunculin B does not interfere with embryonic development but impairs filopodia formation in ISVs, resulting in slower directional migration and failure to form the DLAV (3), phenocopying the migratory behavior of aplnrb mutant embryos (fig.S8, A and B).Notably, even a lower dose of latrunculin B (125 nM) affected aplnrb heterozygous embryos more severely than wild-type siblings (fig.S8, C and D), suggesting a regulatory role of the Apelin pathway in actin dynamics.
Because we found that Apelin signaling is specifically required for the formation of the long filopodia, we next investigated the nature of these filopodia.Notably, we found that tip cells elongated through widening of the dominant filopodium by membrane ruffling (Fig. 5A and movie S4).These observations are reminiscent of the long, Arp2/3 complex-dependent lamellipodium-like protrusions (LLPs) observed in mice (34).To understand the transition from a dominant filopodium to cell elongation, we generated a transgenic line that expresses an ARPC1B-mVenus fusion protein in ECs (Fig. 5B and fig.S9, A and B).Validation of ARPC1B-mVenus localization matched published data on the Arp2/3 complex, demonstrating ARPC1B-mVenus localization in lamellipodia and cell-cell contacts (fig.S9A).Time-lapse imaging revealed Arp2/3 localization to long filopodia in tip cells, followed by membrane ruffling (fig.S9B, arrows, and movie S5), but not to shorter, transient filopodia (fig.S9B, arrowheads, and movie S5).These findings indicate the transition of long filopodia into LLPs before cell elongation.Because aplnrb mutants do not form long filopodia, we were not able to detect if Apelin signaling directly controls Arp2/3 localization in long filopodia (Fig. 5B).To analyze the function of the Arp2/3 complex during tip cell elongation, we treated embryos with the Arp2/3 inhibitor CK666 at 23 hpf, shortly before the initiation of cell elongation (fig.S9, C and D).We found that embryos treated with CK666 exhibited truncated ISVs, confirming the essential role of Arp2/3 in tip cell elongation.
To gain deeper insights into the phenotype of aplnrb mutant embryos, we analyzed time-lapse videos of sprouting ISVs in Tg(fli1a:GAL4FF); Tg(UAS:LIFEACT-EGFP); Tg(kdrl:NLS-mCherry) embryos.In aplnrb mutants, we observed protrusions-albeit at a later time point.However, these protrusions retracted (Fig. 5, C and  D).To determine if tip cells actively retract protrusions, we imaged tip cells in embryos expressing GFP-myosin II in the vasculature [Tg(kdrl:myl9a-EGFP)].Elongating tip cells of wild-type embryos showed myosin localization at the base of filopodia and the cell cortex (Fig. 5, E and E′).Conversely, aplnrb mutant embryos exhibited high myosin enrichment in protrusions during retraction (Fig. 5, E and E″; fig.S10, A to C; and movie S6).
In summary, our results show that Apelin signaling in tip cells is required for the formation of long filopodia that widen to allow rapid cell elongation.This might, in turn, require Arp2/3-dependent actin polymerization.In addition, loss of Apelin signaling leads to mislocalization of myosin to the dorsal protrusions, leading to filopodia retraction by actomyosin contraction.

Apelin signaling induces tip-stalk cell asymmetry
Tip cells undergo asymmetric divisions during their migration, which maintains a larger tip cell size and high VEGF signaling after division (10).By analyzing time-lapse videos of tip cell divisions in Tg(kdrl:Hsa.HRAS-mCherry); Tg(kdrl:NLS-mCherry) double transgenic embryos, we observed a cell size asymmetry after cell division of control tip cells (Fig. 6A).Notably, tip cells in aplnrb mutant embryos underwent symmetrical cell division (Fig. 6A).To quantify the volume of tip cells, we used a mosaic labeling strategy and labeled single tip cells by nuclear mTagBFP-nls and membrane-localized EGFP-CAAX (Fig. 6B).We found that the volume of nondividing tip cells in both wild-type and aplnrb mutant embryos increased over time (fig.S11, A and B).However, the volume of tip cells in aplnrb mutant embryos was smaller on average at both 25 and 30 hpf (fig.S11, A and B).Using our mosaic labeling strategy, we further investigated the relative volume of daughter cells after tip cell division between 25 and 30 hpf.Consistent with a previous report (10), tip cells in wild-type embryos exhibited an asymmetric daughter cell volume after division, resulting in larger daughter tip cells (Fig. 6, C and D).In contrast, division of tip cells in aplnrb mutants caused daughter cells of even size, and occasionally, the daughter tip cells were even smaller than the daughter stalk cells (Fig. 6, C and  D).Asymmetric tip cell division leads to asymmetric partitioning of mRNA, such as kdrl mRNA, and higher ERK activity in daughter tip cells compared to daughter stalk cells (10,35).To determine if the symmetric division of tip cells in aplnrb mutant embryos affect ERK activity, we analyzed cell divisions using confocal time-lapse videos of the transgenic ERK activity reporter Tg(fli1aep:ERK-kinase translocation reporter (KTR)-Clover), which has been used to assess the dynamic Erk activity in ECs (Fig. 6, E and F) (35).The ERK-KTR-Clover is a synthetic reporter that enables quantification of Erk phosphorylation events in cells based on the nucleocytoplasmic ratio of the fluorescent fusion protein (36).After phosphorylation by ERK, the reporter gets exported from the nucleus, meaning that a lower nuclear signal equals higher ERK activity.In line with previous findings (35), we observed asymmetric ERK activity after cell division in daughter tip cells and daughter stalk cells in control embryos (Fig. 6, E and F′).In contrast, we observed similar ERK activity after cell division between daughter tip and daughter stalk cells in aplnrb mutants (Fig. 6, E and F′), suggesting that higher ERK activity in daughter tip cells depends on the cell size asymmetry regulated by Apelin signaling.Considering that tip and stalk cells dynamically compete for the tip cell position during angiogenic sprouting (2), we investigated if Apelin signaling maintains the migratory advantage of tip cells.We observed that loss of aplnrb lead to a dynamic shuffling of tip and stalk cells, a phenotype rarely observed in wild-type embryos (Fig. 6, G and H).We hypothesize that the shuffling of tip cells and stalk cells in aplnrb mutants might be promoted by the symmetric cell divisions.However, we also observed dynamic tip cell shuffling without tip cell divisions (Fig. 6G and movie S7).
These results suggest that the Apelin signaling pathway is an instructive signal for asymmetric daughter cell size after tip cell divisions.As a result, ERK activity in tip cells remains higher than in stalk cells even during cell division, thereby provides tip cells with the migratory advantages necessary to prevent tip-stalk cell shuffling.

Apelin signaling drives sprouting via PI3K and ERK signaling in vivo
The Aplnr binds the G protein subunit Gαi and activates downstream signaling pathways such as PI3K and ERK (37).To determine if Apelin signaling regulates ISV sprouting through these downstream effectors, we used biosensors and pharmacological inhibitors.To visualize PI3K activity in ECs in vivo, we generated a novel transgenic line [Tg(fli1a:PH-AKT-EGFP)] expressing the pleckstrin homology (PH) domain of AKT fused to EGFP, which is commonly used to visualize the localization of phosphatidylinositol 3,4,5-trisphosphate (PIP3) (38), the product of PI3K in ECs (Fig. 7A and fig.S12, A and B).We observed PH-AKT-EGFP localization at the leading edge of the elongating (fig.S12, A and B, and movie S8)

T i p S t a l k T i p S t a l k aplnrb mu281/mu281
Nuclear ERK-KTR (normalized to predivision) Rratio tip/stalk postdivision nuclear ERK KTR  Because GPCRs regulate the β and γ catalytic subunits of class I PI3K (pik3cb and pik3cg in zebrafish), we investigated involvement of these subunits in ISV development.We treated embryos from 20 to 42 hpf with a low dose of the PI3Kγ-specific inhibitor AS-605240 that did not cause severe angiogenic defects in wild-type embryos (fig.S13A) and found that, like the global PI3K inhibitor LY294002, treatment with AS-605240 impaired sprouting in aplnrb heterozygous embryos more severely than in their wild-type siblings (fig.S13, A  and B).To further assess the potential roles of pik3cg and pik3cb, we used a genetic approach by injecting three crRNAs targeting different exons (39,40) of each gene into Tg(fli1a:EGFP) embryos and analyzed the F0 crispants at 32 hpf (fig.S13, C and D).We found that most control embryos formed the DLAV, while 62.8% of embryos injected with crRNAs targeting pik3cg and 47.8% of embryos injected with crRNAs targeting pik3cb fail to form the DLAV (fig.S13, C and D), suggesting that pik3cg and pik3cb may play a role in ISV sprouting.
Given that stimulation of Aplnr-expressing cells with Apelin causes phosphorylation of ERK in vitro (41) and considering the crucial role of ERK activity in ISV development (42), we analyzed ERK activity during vessel sprouting in aplnrb mutants (Fig. 7, E and F).To track dynamic changes in ERK activity in ISVs over time, we recorded time-lapse videos of the ERK kinase translocation reporter Tg(fli1aep:ERK-KTR-Clover) (35,36) from 24 to 30 hpf (before, during, and after cell elongation) in wild-type and aplnrb mutant embryos (Fig. 7, E and F).In wild-type embryos, ERK activity was highest during tip cell elongation at 27 hpf (Fig. 7, E, E′, and F).ERK activity was significantly lower in aplnrb mutant embryos (Fig. 7, E,  E′, and F).At 30 hpf, the tip cells of wild-type embryos reach the dorsal side of the embryo and begin to form the DLAV.Consistent with previous reports, we observed that ERK activity decreased at this developmental time point (35) and became comparable to that of aplnrb mutant embryos (Fig. 7, E and F).To test if the reduced ERK activity is a direct or an indirect consequence of the cell extension-actin dynamics phenotype of apln mutants, we treated Tg(fli1aep:ERK-KTR-Clover) embryos with latrunculin B or CK666 from 24 to 27 hpf and measured ERK activity of tip and stalk cells at 27 hpf (fig.S14).We found that inhibiting actin polymerization and cell elongation equalized the ERK activity of tip and stalk cells in both latrunculin B-treated and CK666-treated embryos (fig.S14, A and B).Therefore, the altered cell shape in aplnrb mutants may contribute to the reduced ERK activity that we observed in tip cells.
Our findings strongly implicate that PI3K and ERK signaling are downstream effectors of Apelin signaling in sprouting ISVs, and we propose a model by which neurovascular Apelin signaling drives tip cell function by modulating PI3K and ERK activity (Fig. 8).

DISCUSSION
Previously, we found that Apelin signaling promotes a proangiogenic state in sprouting ECs and is repressed by Notch signaling (11).Yet, these findings only partially explain the severe angiogenic defects observed in apln and aplnrb mutants.Therefore, in this study, we further investigated how Apelin signaling regulates EC dynamics during angiogenic sprouting.
Apelin is known to be highly expressed in tip cells (11,20), suggesting its potential autocrine function during angiogenesis.However, our findings indicate that paracrine Apelin from neural progenitor cells attracts tip cells during angiogenesis in the zebrafish trunk.Neural progenitor cell-derived Apelin controls the dorsal migration of tip cells and the formation of the DLAV.To form a T-shape and subsequently the DLAV, tip cells might require Apelin signals from anterior and posterior to provide directional information.While ubiquitous and vascular reexpression of Apelin partially rescued ISV sprouting, it did not rescue DLAV formation.One possible explanation could be the lack of directional information due to ubiquitous and vascular reexpression, which might be required for the formation of the DLAV but not ISV.Therefore, a possible function of Apelin as a motogen could stimulate tip cell migration during ISV formation independent of possible Apelin gradients.A possible function as a motogen has already been reported during gastrulation for the second Aplnr ligand Apela (21).Furthermore, tip cells could be able to self-generate an Apelin gradient (43).Because vascular apln expression becomes prominent only after the DLAV has formed, this suggests that Apelin derived from ECs may play a role in the remodeling of the DLAV or signal to other cells in close proximity.However, because we observe aplnrb expression in our reporter lines mainly in ECs, a possible angiocrine function of vascular-derived Apelin is low but possible.Another possibility is that vascular Apelin is rather a marker of active ECs and has only a minor function on the ECs.
By analyzing the migration patterns of angiogenic sprouts in the zebrafish trunk, we identified four characteristic morphological stages that tip cells go through.Consistent with a previous report (3), we detected the formation of long and dominant filopodia toward the dorsal side of the embryo.Apelin signaling is required for the formation of the long filopodia and subsequent elongation of tip cells.Treatment of embryos with latrunculin B to block filopodia formation slows tip cell migration, especially above the horizontal myoseptum (3) and both latrunculin B-treated and apln(rb) mutant embryos fail to form the DLAV, even when the sprouts reach the dorsal side of the embryo (3,11), suggesting that Apelin regulates Actin dynamics in tip cells.We found that the long filopodia, which we observed in our study, are similar to specialized protrusions known as dactylopodia in mice, which evolve from filopodia and rely on the activity of the Arp2/3 complex (34).Consistently, we observed membrane ruffling and localization of Arp2/3 within the most prominent long filopodium, which then expanded, facilitating the tip cell's forward movement.This similarity suggests a conserved mechanism across species and underscores the pivotal role of the Arp2/3 complex not only in lamellipodia formation but also in driving cellular migration through structural changes in protrusions.Our observation that tip cells achieve more efficient migration through the employment of long filopodia indicates that these specialized protrusions are highly effective in facilitating cell migration and represents a novel and unique way of cell migration.Dactylopodia formation is counteracted by myosin II activity in tip cells, which promotes smaller filopodia (34).The enrichment of myosin at the leading edge of tip cells in aplnrb mutant embryos may explain the absence of a dominant filopodium, although smaller filopodia are still present, suggesting that myosin II activity counterbalances the formation of larger, more effective protrusions for migration.Together, our findings imply that Apelin signaling plays a critical role in regulating actin dynamics, triggering the formation of dactylopodia and elongation of tip cells.The interplay of Apelin signaling, myosin II, and the Arp2/3 complex controls the morphological changes required for effective cell migration and provides insights into a sophisticated regulatory mechanism that supports cell migration and vascular morphogenesis.
Our findings position Apelin signaling as an important mediator of sprouting angiogenesis.While loss of Apelin signaling causes severe tip cell defects, it does not cause a complete absence of tip cells or sprouting angiogenesis, as it is the case with the loss of VEGF (4,5).However, Apelin signaling appears to be able to steer sprouting tip cells and regulate tip cell filopodia formation.If Apelin-and VEGF-dependent filopodia (1) are the same type of filopodia requires further research.
In sprouting ECs, VEGF is the main activator of PI3K and MAPK signaling pathways, and loss of VEGF signaling abrogates phosphorylation of ERK (42,44,45).The Aplnr is known to activate PI3K and MAPK signaling pathways in vitro (37,41).Thus far, an in vivo characterization of the pathways modulated by Apelin signaling in ECs was lacking.We found that Apelin signaling regulates tip cell behaviors by modulating PI3K and ERK activity, two well-known and important drivers of sprouting angiogenesis (42,44,46,47).Notably, ERK activity has been shown to be higher in tip cells compared to stalk cells (10,42,48), partially through the asymmetric partitioning of kdrl mRNA in tip cells after cell division (10).Leveraging advancements in ERK activity sensors (35,36), we unveiled a critical role for Apelin signaling to maintain high ERK activity in tip cells during sprouting and after tip cell division.However, because inhibition of actin polymerization and cell elongation leads to equal ERK activity in tip and stalk daughter cells, this suggests that Apelin signaling indirectly modulates ERK activity in tip cells through changes in the cytoskeleton and cell shape.
Previously, we have shown that Apelin signaling regulates c-MYC expression in ECs in vitro (11).Furthermore, other studies reported that c-MYC expression is downstream of the PI3K-AKT-FOXO1 axis (51) and FOXO1 phosphorylation is regulated by Apelin (52).This suggests that Apelin signaling may regulate c-MYC expression through the PI3K-AKT-FOXO1 pathway.However, a recent study suggests that high VEGF-Notch signaling inhibit c-MYC expression, which is required during artery formation (53).This Notch-mediated regulation may balance tip cell proliferation during sprouting, facilitating tip cells to grow and stretch over long distances.
Asymmetric cell division can result in daughter cells with different fates or different sizes (54).Cell size asymmetry of daughter cells has been shown during the division of vascular tip cells (10).However, no signaling pathway controlling this process has yet been identified.Here, we identified Apelin signaling to be required for asymmetric daughter cell size after tip cell divisions.In Caenorhabditis elegans, it was shown that asymmetric cell division relies on the G protein αi subunit acting as a polarity cue to generate differential pulling forces (55).Because the Aplnr couples to the G protein αi subunit, one might speculate that the G protein αi subunit downstream of the Aplnr regulates asymmetric tip cell divisions.Loss of the asymmetric division, which we found to maintain tip cell size, morphology, and ERK activity in agreement with the literature (10), likely further exacerbates the tip cell phenotype in aplnrb mutants.
The similarity of the signaling cascades activated by Apelin and VEGF signaling pathways supports the notion that these pathways cooperatively regulate angiogenesis.Unlike VEGF signaling, overactivation or loss of Apelin signaling, as shown here or in our previous study (11), does not cause ectopic sprouting of the ISVs.This suggests that Apelin signaling mainly adjust rather than induce sprouting.Thus, Apelin signaling may act as an enhancer, finetuning ERK and PI3K activity, as well as dactylopodia-like protrusions in tip cells.As a result, tip cells with high Apelin signaling outcompete ECs with lower Apelin signaling (11).Loss of Apelin signaling equalizes these differences between tip and stalk cells, resulting in tip-stalk cell shuffling.
In summary, our findings reveal a previously unexplored neurovascular cross-talk, showing that neural progenitor cell-derived Apelin coordinates endothelial sprouting and tip cell functionality.

Zebrafish husbandry
All zebrafish housing and husbandry were performed under standard conditions in accordance with institutional [University of Marburg (UMR)] and national ethical and animal welfare guidelines approved by the ethics committee for animal experiments at the Regierungspräsidium Gießen, Germany, as well as the Federation of European Laboratory Animal Science Associations (FELASA) guidelines (56).Embryos were staged by hpf at 28.5°C (57).The following lines were used (see Table 1).

Generation of transgenic lines
To generate the apln BAC construct, we used the published BAC clone from TgBAC(apln:EGFP) bns157 (11).All recombineering steps were performed as described by Bussmann and Schulte-Merker (58), with the modifications as described by Helker et al. (59).The following homology arms were used to generate the targeting polymerase chain reaction (PCR) product of the Venus-pest_Kan cassette: apln_ HA1_Venus_fw: CCACTACAGTATATCAGCTAGCGACTGGCA GGGAAACGGAGGGGAGAGCAACCATGGTGAGCAAG-GGCGAGGAG and apln_HA2_kanR_rev: CACAGCAGAGAAAC-CACCAGCACAATCACCAGCGTCAAGATCTTCACATTAC-CATGGAGAAGTTACTATTCCG.The kanamycin cassette was removed with a flippase.

Confocal microscopy
Zebrafish embryos were mounted in 1% low melt or 0.3% normal agarose.E3 and agarose were supplemented with tricaine (19.2 mg/ liter).If imaged later after 30 hpf, then embryos were treated with 0.1% (w/v) propylthiouracil from 24 hpf.Fluorescence images were acquired on a Leica Stellaris 8 confocal microscope equipped with HC FLUOTAR L VISIR 25x/0.95WATER and HC PL APO CS2 40x/1.10WATER objectives and an Oko-lab incubator set to 28.5°C for time-lapse experiments.

Filopodia quantification
Filopodia were quantified from maximum intensity projections of confocal stacks acquired from transgenic Tg(kdrl:EGFP-CAAX) embryos.Siblings are heterozygous or wild-type for aplnrb, and mutants are homozygous (aplnrb mu281/mu281 ).Length and the corresponding angle relative to the DA for each filopodium adjacent to the leading edge of the tip cells were measured in Fiji.Filopodia with an angle of 45° to 90° relative to the DA were classified as dorsal, and filopodia with an angle of 0° to 45° were classified as lateral filopodia.

Quantification of protrusion retractions
Protrusions formed or retracted by tip cells (excluding filopodia) were observed between 23 and 30 hpf for wild-type or drug-treated embryos or due to delayed protrusion formation between 30 and 39 hpf for aplnrb mu281/mu281 embryos.Same control embryos were used in Fig. 5D and fig.S12D.

Line graph intensity measurements
Line graphs of fluorescence intensity [for Tg(kdrl:myl9a-EGFP) ip5Tg or Tg(fli1a:PH-AKT-EGFP) mr23 ] were generated by measuring the fluorescence intensity (in confocal maximum intensity projections) from the leading edge of the tip cell toward the center of the cell in Fiji.The values for each distance were then normalized to the average intensity across the whole line.

Cell tracking
The migration of tip cells was measured by using the spots function in Imaris 9.7.2.The linear distance in dorsal direction between the leading edge of the tip cell (excluding filopodial protrusions) and the aorta was measured (dorsal distance).

Mosaic tip cell labeling
To label individual tip cells, a plasmid was generated that expresses nuclear BFP in one and GFP-CAAX in the other direction driven by a bidirectional fli1a promoter (BFP-nls:fli1a:EGFP-CAAX) (65).The construct was injected in the offspring of an aplnrb wt/mu281 Tg(kdrl:Hsa.HRAS-mCherry) s896 incross at one-cell stage.Embryos were imaged the next day at 25 and 30 hpf.
Cell volumes were measured in Imaris 9.7.2 by generating a surface of tip cells positive for the GFP-CAAX signal.Cell volumes were normalized to sibling cell size at 25 hpf.

Tg(hsp70:apln) overexpression experiments
For the temporal ubiquitous overexpression of Apelin via the Tg(hsp70:apln) mu269 transgene, apln wt/mu267 Tg(hsp70:apln) mu269 fish were outcrossed to apln wt/mu267 fish that do not carry the heat shock transgene.The offspring was then subjected to a single heat shock at 37°C for 60 min at 24 hpf and imaged under the confocal microscope at 32 hpf.

Quantification of ERK activity in vivo
Nuclear signal of the ERK activity reporter [Tg(fli1a:ERK-KTR-Clover)] was measured in Imaris 9.7.2 as described in (35).Two to five tip cells of adjacent ISVs above the yolk extension were analyzed per embryo.Intensities were normalized to the average nuclear ERK intensity of the aorta section adjacent to the analyzed ISVs.To generate the color-coded nuclei, the Clover channel was masked with the generated surface, transferred to Fiji, and converted to an 8-bit image and the Lookup Table (LUT) was set to "16 colors." To measure ERK activity after cell division, confocal time-lapse videos of Tg(fli1a:ERK-KTR-Clover) of aplnrb mutant embryos or wild-type embryos were analyzed between 25 and 27 hpf.Nuclear intensity of the ERK reporter was measured (as described above) 35 to 45 min after tip cell division.

crRNA design
To generate P0 knockouts of the pik3cg and pik3cb genes, three synthetic CRISPR RNAs (crRNAs) targeting different exons were annealed to trans-activating RNA (trRNA) and coinjected into one-cell stage embryos with Cas9 protein as described previously (40).Cas9 protein without crRNA-trRNA was used as a control.
Before the experiments, functionality of the crRNAs was tested by HRM analysis.Synthetic RNA and Cas9 protein were purchased from IDT. Predesigned crRNAs were chosen if available; otherwise, custom crRNAs were designed with the IDT web tool.The crRNAs were selected based on highest on/off-target scores.The following crRNAs were used (see Table 2).
For "low-dose treatments, " a dose of the respective inhibitor that did not cause an obvious phenotype in wild-type embryos was determined.Next, aplnrb wt/mu281 Tg(fli1a:EGFP) y1 fish were outcrossed to wild-type fish and the offspring treated from 22 to 42 hpf.Embryos were analyzed under a fluorescence microscope and grouped into different categories (see Quantification of phenotypes) and genotyped.

Quantification of phenotypes
For every embryo, somites 5 to 15 were analyzed: normal (no phenotype): 10 fully developed ISVs and connected DLAV; mild: ISVs fully outgrown but 1 to 5 gaps in the DLAV; medium: ISVs fully outgrown but more than 5 gaps in the DLAV; and severe: 1 to 6 ISVs shortened/truncated.

Statistics and reproducibility
Statistical analysis was performed in Prism 9 (GraphPad).Respective tests and n numbers are indicated in the figure legends.For normally distributed data, we used two-tailed unpaired Student's t test to compare between two means and one-way analysis of variance (ANOVA) with Tukey's multiple comparison test for comparison of multiple conditions.For non-normally distributed data, Mann-Whitney test was used to compare between two means and one-way ANOVA with Kruskal-Wallis test was used for comparison of multiple conditions.For grouped data, two-way ANOVA with Tukey's multiple comparison test or ANOVA with Bonferroni correction (when comparing selected values of multiple conditions, e.g., two conditions over time; Fig. 7F) was used.Values are shown as mean ± SD.P values of <0.05 were deemed as significant.
and T-shaped (fig.S12B) tip cells.Intriguingly, aplnrb mutants did not display PH-AKT-EGFP localization toward the leading edge; instead, the PH-AKT-EGFP signal was more evenly distributed throughout the cell (Fig.7, A and B).To investigate a possible role of PI3K downstream of Apelin signaling, we treated aplnrb heterozygous embryos from 20 to 42 hpf with a low concentration of the PI3K inhibitor LY294002 that did not cause severe angiogenic defects in wild-type embryos (Fig.7, C and D).Notably, heterozygous aplnrb mutant embryos treated with the PI3K inhibitor displayed a stronger phenotype compared to wild-type siblings (Fig.7, C and D).Confocal time-lapse imaging revealed that protrusions retract in embryos treated with a higher dose of the PI3K inhibitor (25 μM LY294002), similarly to what we observed in aplnrb mutant embryos (fig.S12, C and D).